Citrate-reduced Silver Colloids Research Articles

Characterization of Bacteria Using Surface-Enhanced Raman Spectroscopy (SERS): Influence of Microbiological Factors on the SERS Spectra.

SERS is currently being explored as a rapid method for identification of bacteria but variation in the experimental procedures has resulted in considerable variation in the spectra reported for a range of bacterial species. Here, we show that mixing bacteria with a conventional citrate-reduced silver colloid (CRSC) and drying the resulting suspension yield highly reproducible spectra. These signals were due to intracellular components released when the structure of the bacteria was disrupted during sample preparation. This reproducibility allowed us to examine the effects of variables that do not arise in SERS of simple solutions but are relevant in studies of bacteria. These included growth phase and biological variation, which occurred when the same bacterial isolates were cultured under nominally identical conditions on different days. It was found that even under optimal standardized conditions the effect of differences in experimental parameters such as growth phase was very large in some bacterial species but insignificant in others. This suggests that it is important to avoid drawing general conclusions about bacterial SERS based on studies using small numbers of samples. Similarly, discrimination between bacterial species was straightforward when a small number of isolates with distinct spectral features were investigated; however, this became more challenging when more bacterial species were included, as this increased the possibility of finding different species of bacteria with similar spectra. These observations are important because they clearly delineate the challenges that will need to be addressed if SERS is to be used for clinical applications.

Detection and characterisation of bacteria causing lung infection in people with Cystic Fibrosis (CF) by surface-enhanced Raman spectroscopy (SERS)

Rapid and accurate identification of pathogens in CF could ensure prompt treatment with the most appropriate antibiotic; potentially improving outcomes and shortening hospital stays. As traditional culture methods for detecting bacteria are time-consuming there is a growing interest in SERS as a novel culture-free technique that produces a whole-organism spectroscopic fingerprint at high speed. Bacterial isolates including Pseudomonas aeruginosa (n=32), Staphylococcus aureus (n=5), Streptococcus pneumoniae (n=5) were incubated for 6 h at 37 °C/180 r.p.m., with a starting optical density (OD) of 0.15. After adjustment of the OD to 0.3, bacterial cells were harvested by centrifugation at 9000 rcf for 3 min and washed three times with dH2O. Bacterial pellets were mixed with citrate reduced silver colloid (CRSC) and dried. Spectra were recorded (4×10 s at 785 nm) and analysed within GRAMS/Al using Principal Component Analysis (PCA). Spectra of P. aeruginosa isolates (n=32) were separated into two distinct groups; the spectra of one group (n=12) was dominated by the pigment pyocyanin with vibrational brands present at 1350, 1492, 1598 and 1615 cm-1. The other group (n=20) had characteristic vibrational bands at 661, 735 and 800 cm-1 which correspond to guanine, adenine and uracil, respectively. S. aureus has a main characteristic band at 735 cm-1 and S. pneumoniae has a characteristic band present at 480 cm-1. Bacterial species clustered separately when analysed by PCA. Reproducible and distinguishable SERS spectra of bacterial isolates were obtained, and it was possible to differentiate between different bacterial species using PCA. These results suggest SERS has the potential to rapidly detect bacteria.

Quantitative surface‐enhanced resonance Raman scattering analysis of methylene blue using silver colloid

AbstractQuantitative analysis of methylene blue (MB) has been performed with Surface Enhanced Raman Scattering, SERS, using citrate reduced silver colloid activated with NaCl. In this study, the combination of SERS occurring at the proximity of the plasmon surface and of resonance Raman with excitation wavelength matched to the maximum absorbance of the molecule being analyzed, was successfully applied to maximize the Raman scattering intensity. The surface‐enhanced resonance Raman scattering (SERRS) spectra of aqueous solutions of the dye at various concentrations were collected in right‐angle scattering geometry configuration which results favorable when nanocolloidal dispersions are used. The MB concentration was correlated with the intensity of the peak centered at 1,625 cm−1 in the SERRS spectrum and this correlation resulted in an extremely sensitive quantitative investigation of the MB with limit of detection in pM level. Among others, this method can be easily applied for the examination of both the selectivity of waste water purification membranes for relevant small molecular weight contaminants or/and the solar photocatalytic effectiveness of such membranes for the degradation of pertinent pollutants by their quantitative assessment in the permeate at very low concentration range.

Optimization of parameters for histamine detection in fish muscle extracts by surface‐enhanced Raman spectroscopy using silver colloid SERS substrates

This study was focused on investigating possibility of histamine detection in fish by surface‐enhanced Raman spectroscopy (SERS). Four different silver colloids were used as SERS substrates in combination with different aggregation agents. Excitation wavelength, aggregating agent concentration and time of data acquisition have been optimized in order to obtain maximum enhancement of histamine signals. Best results were obtained with 514.5‐nm excitation wavelength, citrate reduced silver colloid and 0.23 mol/l NaBH4 as aggregating agents enabling detection of histamine in water solutions at concentration of 5 × 10−6 mol/l. Calibration curve was constructed by applying Langmuir adsorption isotherm formula to fit the experimental data. Because of interferences from other components, histamine was detected at concentration of 1 × 10−4 mol/l (111.2 mg/kg of fish) in fish muscle extracts. Although further improvements are needed to achieve detection and quantification of histamine at legislative set limits these results show a potential for development of new histamine detection method in fish and fish products. Copyright © 2016 John Wiley & Sons, Ltd.

SERS Discrimination of Closely Related Molecules: A Systematic Study of Natural Red Dyes in Binary Mixtures

Recent methodological and instrumental advances have afforded surface-enhanced Raman spectroscopy (SERS) wide recognition in the cultural heritage field as a powerful tool for the analysis of organic colorants. The many advantages of SERS are counterbalanced by the fact that it has limited ability to resolve dye mixtures. In this work, a systematic study was conducted to determine the technique’s ability to discriminate closely related molecules in binary mixtures for a selection of natural red dyes widely used in painting and textile dyeing (alizarin, purpurin, carminic and laccaic acids, brazilein). Spectra were recorded on two different metal substrates, i.e., citrate-reduced silver colloids and silver films over nanospheres (AgFONs). After working on reference systems, the study was expanded to include analysis of red lake oil paint reconstructions upon hydrolysis with hydrofluoric acid, aiming to gain a deeper understanding of how varying the experimental conditions may affect dye identification. It ...

Adsorption mechanisms of RNA mononucleotides on silver nanoparticles

Surface-enhanced Raman scattering (SERS) of four RNA mononucleotides (AMP, GMP, CMP and UMP) has been studied on the citrate-reduced silver colloid aggregated with sodium sulfate. Concentration dependent spectra in the range of 1×10−7–1×10−4moldm−3 were obtained, assigned and interpreted according to the surface selection rules. For purine mononucleotides, AMP and GMP, adsorption onto the silver nanoparticles through the six-membered ring of the nitrogenous base was suggested. Concentration dependent splitting of the ring breathing band in the spectra of AMP indicated coexistence of two species on the silver surface, which differed in contribution of the adenine N1 atom and the exocyclic NH2 group in binding. Unlike the AMP spectra, the spectra of GMP implied only one mode of adsorption of the molecules onto the silver nanoparticles, taking place through the guanine N1H and CO group. Weak SERS spectra of pyrimidine mononucleotides, CMP and UMP, pointed to involvement of carbonyl oxygen in adsorption process, whereby the molecules adopted the position on the nanoparticles with ribose close to the metal surface. Intense bands in the low wavenumber region, associated with stretching of the formed AgN and/or AgO bonds, supported chemical binding of the RNA mononucleotides with the silver surface.

SERS-active silver colloids prepared by reduction of silver nitrate with short-chain polyethylene glycol

We report a fast, one-step, facile, and green preparation method that yields very stable and biocompatible silver colloids that are highly active as surface-enhanced Raman spectroscopy (SERS) platforms that has a possible application in biomedicine. Reduction of silver nitrate has been carried out using polyethylene glycol (PEG) which acts as both reducing agent and stabilizer. It turned out that the -OH groups provided by the addition of NaOH represent a key element in the successful synthesis of PEG-coated silver nanoparticles (AgNPs). The as-obtained silver colloids have been characterized by UV-visible spectroscopy, transmission electron spectroscopy, and SERS using 532- and 633-nm laser lines on a dispersive Raman spectrometer. Several analytes as methylene blue, p-aminothiophenol, amoxicillin, and Cu(PAR)2 were used to prove SERS enhancement of the obtained silver colloid. It has been found that the PEGylated AgNPs provide SERS signals comparable to those achieved using classical hydroxylamine and citrate-reduced silver colloids, thus demonstrating the ability of this new method to prepare biocompatible silver colloids.

Near-Infrared Surface-Enhanced Raman Spectroscopy (NIR-SERS) for the Identification of Eosin Y: Theoretical Calculations and Evaluation of Two Different Nanoplasmonic Substrates

This work demonstrates the development of near-infrared surface-enhanced Raman spectroscopy (NIR-SERS) for the identification of eosin Y, an important historical dye. NIR-SERS benefits from the absence of some common sources of SERS signal loss including photobleaching and plasmonic heating, as well as an advantageous reduction in fluorescence, which is beneficial for art applications. This work also represents the first rigorous comparison of the enhancement factors and the relative merits of two plasmonic substrates utilized in art applications; namely, citrate-reduced silver colloids and metal film over nanosphere (FON) substrates. Experimental spectra are correlated in detail with theoretical absorption and Raman spectra calculated using time-dependent density functional theory (TDDFT) in order to elucidate molecular structural information and avoid relying on pigment spectral libraries for dye identification.

Analysis of 5-Hydroxyisoflavones by Surface-Enhanced Raman Spectroscopy: Genistein and Methoxy Derivatives

A series of 5-hydroxy-isoflavones-genistein, biochanin A, prunetin, and 4',7-dimethoxygenistein-have been studied by surface-enhanced Raman spectroscopy (SERS). Citrate reduced silver colloids were employed as a standard technique to measure SER spectra over a range of pH and concentrations. Density functional theory calculations were used to assist in determining the mode of interaction of isoflavones with the silver nanoparticles. It is revealed that biochanin A and prunetin interact with the silver nanoparticles upon deprotonation of the 7- and the 4'-OH groups, respectively, to show SERS activity. Correlations of their spectra with SERS of genistein strongly support the presence of multiple interaction modes involving both of the OH groups in genistein, in a similar manner to daidzein. Surprisingly, however, under these conditions, the 5-OH group was found to be noninteractive as revealed by attempts to measure SERS of 4',7-dimethoxygenistein. This was attributed partly to the low solubility and, more importantly, to the influence of steric hindrance, caused by the position of the pendant phenyl ring, which prevented interaction with the Ag colloid surface. These results complement recent work on daidzein and formononetin and provide further insight into understanding the SER spectra of isoflavones.

Optimization of silver nanoparticles for surface enhanced Raman spectroscopy of structurally diverse analytes using visible and near-infrared excitation

Several experimental parameters affecting surface enhanced Raman (SER) signals using 488, 785 and 1064 nm excitation for eight diverse analytes are reported. Citrate reduced silver colloids having average diameters ranging from 40 ± 10 to 100 ± 20 nm were synthesized. The nanoparticles were characterized by transmission electron microscopy, dynamic light scattering and absorbance spectrophotometry before and after inducing nanoparticle aggregation with 0.99% v/v 0.5 M magnesium chloride. The nanoparticle aggregates and SERS signal were stable between 30 and 90 minutes after inducing aggregation. For the analytes 4-mercaptopyridine, 4-methylthiobenzoic acid and the dipeptide phenylalanine-cysteine using all three excitation wavelengths, the highest surface area adjusted SER signal was obtained using 70 ± 20 nm nanoparticles, which generated 290 ± 40 nm aggregates with the addition of magnesium chloride. The decrease in the SER signal using non-optimum colloids was 12 to 42% using 488 nm excitation and larger decreases in signal, up to 92%, were observed using near infrared excitation wavelengths. In contrast, pyridine, benzoic acid, and phenylalanine required 220 ± 30 nm aggregates for the highest SER signal with 785 or 1064 nm excitation, but larger aggregates (290 ± 40 nm) were required with 488 nm excitation. The optimum experimental conditions measured with the small molecule analytes held for a 10 amino acid peptide and hemoglobin. Reproducible SERS measurements with 2 to 9% RSD have been obtained by considering nanoparticle size, aggregation conditions, excitation wavelength and the nature of the analyte-silver interaction.

Comparative Analysis of Surface-Enhanced Raman Spectroscopy of Daidzein and Formononetin

Phytoestrogens daidzein (4',7-dihydroxy-isoflavone) and formononetin (4'-methoxy-7-hydroxy-isoflavone) have been studied by surface-enhanced Raman (SER) spectroscopy. Spectra were acquired in the presence of citrate-reduced silver colloids, over a range of pH and concentrations. Density functional theory calculations were used to assist assignment of the normal Raman spectra and help determine the mode of interaction of isoflavones with the silver nanoparticles. Formononetin does not show SER activity unless the 7-OH group is deprotonated, and accordingly, the interaction with the silver surface occurs via the deprotonated site. Daidzein, on the other hand, appears to contain multiple species at the surface, interacting via both the hydroxyl groups at 7-OH and 4'-OH, after deprotonation. This is an important result that points to potential future SERS applications in phytoestrogen analysis and provides a foundation for understanding the SER spectra of isoflavones.

Spectroscopic study of surface enhanced Raman scattering of caffeine on borohydride-reduced silver colloids

The surface enhanced Raman scattering (SERS) of caffeine on borohydride-reduced silver colloids system under different aqueous solution environment has been studied in this paper. The relative intensity of SERS of caffeine significantly varies with different concentrations of sodium chloride and silver particles. However, at too high or too low concentration of sodium chloride and silver particle, the enhancement of SERS spectra is not evident. The SERS spectra of caffeine suggest that the contribution of the charge transfer mechanism to SERS may be dominant. The chloride ions can significantly enhance the efficiency of SERS, while the enhancement is selective, as the efficiency in charge transfer enhancement is higher than in electromagnetic enhancement. Therefore, it can be concluded that the active site of chloride ion locates on the bond between the caffeine and the silver surface. In addition, the SERS spectra of caffeine on borohydride-reduced and citrate-reduced silver colloids are different, which may be due to different states caffeine adsorbed on silver surface under different silver colloids.

Quantitative analysis of methyl green using surface-enhanced resonance Raman scattering

Surface-enhanced resonance Raman scattering (SERRS) spectra of aqueous solutions of the triphenylmethane dye methyl green have been obtained for the first time by use of citrate-reduced silver colloids and a laser excitation wavelength of 632.8 nm. Given the highly fluorescent nature of the analyte, which precluded collection of normal Raman spectra of the dye in solution and powdered state, it was highly encouraging that SERRS spectra showed no fluorescence due to quenching by the silver sol. The pH conditions for SERRS were optimised over the pH range 0.5-10 and the biggest enhancement for SERRS of this charged dye was found to be at pH 2.02, thus this condition was used for quantitative analysis. SERRS was found to be highly sensitive and enabled quantitative determination of methyl green over the range 10(-9) to 10(-7) mol dm(-3). Good fits to correlation coefficients were obtained over this range using the areas under the vibrational bands at 1615 and 737 cm(-1). Finally, a limit of detection of 83 ppb was calculated, demonstrating the sensitivity of the technique.

Surface-Enhanced Raman Scattering Spectroscopy of Resveratrol

We report here, for the first time, the surface-enhanced Raman scattering (SERS) spectra of resveratrol using KNO3-aggregated citrate-reduced silver (Ag) colloids. The technique provided a substantial spectral enhancement and therefore good quality spectra of resveratrol at parts per million (ppm) concentrations. The detection limit was found to be <1 μM, equivalent to <0.2 ppm. The SERS profile additionally closely resembled its normal solid-state Raman spectrum with some changes in relative intensity. These intensity changes, together with a precise band assignment aided by density functional theory calculations at the B3LYP/6–31G(d) level, allowed the determination of the structural orientation of the adsorbed resveratrol on the surface of the metal nanoparticles. In particular, the SERS spectra obtained at different resveratrol concentrations exhibited concentration-dependent features, suggesting an influence of surface coverage on the orientation of the adsorbed molecules. At a high concentration, an adoption of close-to-upright orientation of resveratrol adsorbed on the metal surface through the p-OH phenyl ring is favoured. The binding structure is, however, altered at lower surface coverage when the concentration decreases to a tilted orientation with the trans-olefin C=C bond aligning closer to parallel to the surface of the Ag nanoparticles.

Effects and anomalies that can occur in SERS spectra of biological molecules when using a wide range of aggregating agents for hydroxylamine-reduced and citrate-reduced silver colloids

SERS spectra of cysteine and proline were measured for citrate-reduced and hydroxylamine-reduced silver colloids using aggregating agents K 2SO 4, NaNO 3, NaCl, Ca(NO 3) 2, CaCl 2, Na 2SO 4, KNO 3 and KCl, to compare the effects of aggregation on two different colloid environments. High intensity anomalous bands were found for both colloids on the addition of the aggregating agents. On the addition of cysteine these anomalous bands disappeared. However, when proline was analysed, the anomalous bands remained, indicating the importance of characterising the colloidal sol to differentiate between band patterns from the analyte and those from the colloid in the presence of an aggregating agent. K 2SO 4 gave the greatest enhancement (∼10 3) of any aggregating agent for cysteine with the hydroxylamine-reduced colloid, in contrast to the citrate-reduced colloids where similar enhancement factors (∼10 2) were found with several different aggregating agents. Through the analysis of different SERS spectra for amino acids with different aggregating agents and colloids, a better understanding of the SERS technique may be achieved and its application in biology may be expanded.

Potential of Surface-Enhanced Raman Spectroscopy for the Rapid Identification of Escherichia Coli and Listeria Monocytogenes Cultures on Silver Colloidal Nanoparticles

Surface-enhanced Raman (SERS) spectra of various batches of bacteria adsorbed on silver colloidal nanoparticles were collected to explore the potential of the SERS technique for rapid and routine identification of E. coli and L. monocytogenes cultures. Relative standard deviation (RSD) of SERS spectra from silver colloidal suspensions and ratios of SERS peaks from small molecules (K(3)PO(4)) were used to evaluate the reproducibility, stability, and binding effectiveness of citrate-reduced silver colloids over batch and storage processes. The results suggested consistent reproducibility of silver colloids over batch process and also stability and consistent binding effectiveness over an eight-week storage period. A variety of mixtures of E. coli/L. monocytogenes cultures with different colloidal batches revealed that, despite large variations in relative intensities and positions of SERS active bands, characteristic and unique bands at 712 and 390 cm(-1) were consistently observed and were the strongest in E. coli and L. monocytogenes cultures, respectively. Two specific bands were used to develop simple algorithms in the evaluation of binding effectiveness of silver colloids over storage and further to identify E. coli and L. monocytogenes cultures with a 100% success. A single spectrum acquisition took 5 approximately 6 min, and a minimum of 25 microL silver colloid was directly mixed with 25 microL volume of incubated bacterial culture. The short acquisition time and small volume of incubated bacterial culture make silver colloidal nanoparticle based SERS spectroscopy ideal for potential use in the routine and rapid screening of E. coli and L. monocytogenes cultures on large scales. This is the first report of the development of simple and universal algorithms for bacterial identification from the respective exclusive SERS peaks.

Surface-Enhanced Raman Spectroscopy (SERS) for Sub-Micromolar Detection of DNA/RNA Mononucleotides

Surface-enhanced Raman (SER) spectra of all the DNA/RNA mononucleotides have been obtained with high sensitivity using citrate-reduced silver colloids aggregated with MgSO4, rather than the more usual halide ions, which were found to prevent enhancement of these compounds. The SERS spectra of adenine, guanine, thymine, cytosine, and uracil were recorded along with their corresponding nucleosides and 5'-deoxynucleotides. For the cytosine series, all three spectra had similar relative band intensities but the spectra of adenine were different from those of adenosine and dAMP, probably due to differences in orientation on the surface. No enhanced bands from the phosphate or sugar groups were observed. There were general similarities between the SERS spectra of the purine mononucleotides and the pyrimidine mononucleotides, but the spectra were sufficiently different to allow each of them to be distinguished. This method can therefore be used for high sensitivity, label-free identification of mononucleotides.

Surface-Enhanced Raman Spectroscopy as a Probe of Competitive Binding by Anions to Citrate-Reduced Silver Colloids

Citrate-reduced silver colloids (CRSCs) are used extensively for surface-enhanced Raman scattering (SERS) studies of cations but are typically found to be ineffective for detection of anions unless they are treated with compounds that give them positively charged coatings. In this work CRSCs which were suitable for detection of anions were generated by treatment with aggregating agents that did not bind strongly to the silver surface. Under these conditions the major factor determining the enhancement of added anions was their ability to displace whatever anions were already present. In the case of CRSCs, residual citrate was observed when the colloids were aggregated with sulfate salts, since neither sulfate nor the residual nitrate displaced it. On addition of more strongly binding anions, such as halides, the citrate was displaced and the bands of the added analyte appeared, allowing them to be detected without the need for creation of positively charged coatings. It was found that the relative affinities of the anions, as determined by displacement experiments monitored by SERS, followed the solubilities of their silver salts, presumably because both properties are strongly dependent on the strength of the Ag-anion bonds. The relative affinities determine which anions can be detected in the presence of which others; nitrate, sulfate, and perchlorate are lower in the series than citrate and so are not observed. Displacement experiments show that dipicolinic acid (DPA) and Cl(-) have similar (stronger) binding, but they can be displaced in turn by Br(-) and I(-), which have the highest affinity and lowest solubility. This model allows a broad range of previous observations to be rationalized and allows the experimental conditions suitable for detection of particular new analytes to be designed on rational principles.

Quantitative surface-enhanced Raman spectroscopy of dipicolinic acid—towards rapid anthrax endospore detection

Dipicolinic acid (DPA) is an excellent marker compound for bacterial spores, including those of Bacillus anthracis (anthrax). Surface-enhanced Raman spectroscopy (SERS) potentially has the sensitivity and discrimination needed for trace DPA analysis, but mixing DPA solutions with citrate-reduced silver colloid only yielded measurable SERS spectra at much higher (>80 ppm) concentrations than would be desirable for anthrax detection. Aggregation of the colloid with halide salts eliminated even these small DPA bands but aggregation with Na2SO4(aq) resulted in a remarkable increase in the DPA signals. With sulfate aggregation even 1 ppm solutions gave detectable signals with 10 s accumulation times, which is in the sensitivity range required. Addition of CNS- as an internal standard allowed quantitative DPA analysis, plotting the intensity of the strong DPA 1010 cm(-1) band (normalised to the ca. 2120 cm(-1) CNS- band) against DPA concentration gave a linear calibration (R2 = 0.986) over the range 0-50 ppm DPA. The inclusion of thiocyanate also allows false negatives due to accidental deactivation of the enhancing medium to be detected.

Raman, IR, and surface-enhanced Raman spectroscopy of papaverine: An automated setup for in situ synthesis of the silver substrate and recording of the SER spectra

Raman, IR, and surface-enhanced Raman scattering (SERS) were employed for the vibrational characterization of papaverine in the free and adsorbed state. The vibrational assignments were performed by means of density functional theory (DFT) calculations. The adsorption of the molecule was concluded with the isoquinoline and the benzene ring close to the silver surface in tilted orientations. Employing a sequential injection system, an automatized setup was developed for reliable, accurate and rapid recording of FT-SER spectra. The colloidal silver substrate was synthesized in situ in a 2 ml quartz cuvette by reducing silver nitrate with hydroxylamine. The FT-SER spectrum of papaverine obtained with the developed experimental setup exhibits the same characteristics as the SER spectrum obtained with a citrate reduced silver colloid.